Pituitary-tumor-transforming-genes, and related products

ABSTRACT

Polypeptides are expressed by the pituitary-tumor-transforming-gene (PTTG), formerly known as pituitary-tumor-specific-gene (PTSG), and nucleic acids encode them. Examples are the human and rat PTTG proteins. The nucleic acids may be applied to the production of a recombinant protein, and to the detection of the presence of PTTG genes in different species. The nucleic acids may be operatively linked to a vector, optionally provided with control and expression sequences and/or being carried by a host cell. The nucleic acids may also be delivered to a mammal to compensate for the absence, or a defective expression, of endogenous protein. The nucleic acids, proteins, and antibodies are also employed in disgnostic assays, as well as, for example, in the production of anti-PTTG antibodies (protein), therapeutic compositions and other applications of the proteins and antibodies. Various kits utilize nucleic acids, polypeptides, and/or antibodies. A transgenic non-human mammal expresses PTTG.

This Application was filed under 35 U.S.C. § 371, based on internationalapplication PCT/US97/21463, filed Nov. 21, 1997, and claims the priorityof the filing date of U.S. Provisional Application Ser. No. 60/031,338,entitled NUCLEIC ACID ENCODING A FAMILY OFPITUITARY-TUMOR-SPECIFIC-GENES, AND PRODUCTS RELATED THERETO, by ShlomoMelmed and Lin Pei, filed Nov. 21, 1996.

This invention was made at least in part with United States Governmentsupport under Grant Number DK42742, awarded by the National Institutesof Health. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to nucleic acids and proteins encodedthereby. Invention nucleic acids encode a novel family ofpituitary-tumor-specific-gene proteins. The invention also relates tomethods for making and using such nucleic acids and proteins.

2. Description of the Background

Cancers and tumors are the second most prevalent cause of death in theUnited States, causing 450,000 deaths per year. One in three Americanswill develop cancer, and one in five will die of cancer (ScientificAmerican Medicine, part 12, I, 1, section dated 1987). While substantialprogress has been made in identifying some of the likely environmentaland hereditary causes of cancer, the statistics for the cancer deathrate indicates a need for substantial improvement in the therapy forcancer and related diseases and disorders.

A number of cancer genes, i.e., genes that have been implicated in theetiology of cancer, have been identified in connection with hereditaryforms of cancer and in a large number of well-studied tumor cells. Studyof cancer genes have helped provide some understanding of the process oftumorigenesis. While a great deal more remains to be learned aboutcancer genes, the presently known cancer genes serve as useful modelsfor understanding tumorigenesis.

Cancer genes are broadly classified into “oncogenes” which, whenactivated, promote tumorigenesis, and “tumor suppressor genes” which,when damaged, fail to suppress tumorigenesis. While theseclassifications provide a useful method for conceptualizingtumorigenesis, it is also possible that a particular gene may playdiffering roles depending upon the particular allelic form of that gene,its regulatory elements, the genetic background and the tissueenvironment in which it is operating.

Tumor suppressor genes are genes that in their wild-type alleles,express proteins that suppress abnormal cellular proliferation. When thegene coding for a tumor suppressor protein is mutated or deleted, theresulting mutant protein or the complete lack of tumor suppressorprotein expression may fail to correctly regulate cellularproliferation, and abnormal cellular proliferation may take place,particularly if there is already existing damage to the cellularregulatory mechanism. A number of well-studied human tumors and tumorcell lines have been shown to have missing or nonfunctional tumorsuppressor genes. Examples of tumor suppression genes include, but arenot limited to the retinoblastoma susceptibility gen or RB gene, the p53gene, the deleted in colon carcinoma (DDC) gene and theneurofibromatosis type 1 (NF-1) tumor suppressor gene. Loss of functionor inactivation of tumor suppressor genes may play a central role in theinitiation and/or progression of a significant number of human cancers.

Anterior pituitary tumors are mostly benign hormone-secreting ornon-functioning adenomas arising from a monoclonal expansion of agenetically mutated cell. Pathogenesis of tumor formation in theanterior pituitary has been intensively studied. Mechanisms forpituitary tumorigenesis involve a multi-step cascade of recentlycharacterized molecular events. The most well characterized oncogene inpituitary tumors is gsp, a constitutively active Gasα resulting formactivating point mutations in this gene.

Gasα mutations occur in about 40% of GH-secreting tumors, andconstitutively activated CREB is also found in a subset of these tumors.Although the importance of GSα mutant proteins in the development ofgrowth-hormone secreting pituitary tumors is well established, onlyabout one third of these tumors contains these mutations, indicating thepresence of additional transforming events in pituitary tumorigenesis.Although point mutations of Ras oncogene, loss of heterozygosity (LOH)near the Rb locus on chromosome 13, and LOH on chromosome 11 have beenimplicated in some pituitary tumors, the mechanism that causes pituitarycell transformation remains largely unknown. Thus, there is a need inthe art for additional pituitary derived proteins that are associatedwith pituitary cell transformation.

SUMMARY OF THE INVENTION

The present invention relates to isolated, purifiedMammalian-pituitary-Transforming-Gene (PTTG) proteins, formerly namedMammalian Pituitary-Tumor-Specific-Gene (PTSG) proteins. The PTTGproteins of the invention and fragments thereof, are useful inbioassays, as immunogens for producing anti-PTTG antibodies, or intherapeutic compositions containing such proteins and/or antibodies.

This invention also relates to a transgenic non-human mammal thatexpresses PTTG protein.

The present invention also relates to isolated nucleic acids encodingPTTG (PTSG) proteins of mammalian origin, such as human, rat, etc. ThePTTG encoding nucleic acid is also provided in the form of a vectorcarrying it, as hybridizing probes/primers, in host cells carrying them,as anti-sense oligonucleotides, in DNA and RNA forms, and relatedcompositions. The nucleic acid molecules described herein may beincorporated into expression systems known to those of skill in the art.The PTTG nucleic acids are useful as probes for assaying for thepresence and/or amount of a PTTG gene or mRNA transcript in a givensample. The nucleic acid molecules described herein, and oligonucleotidefragments thereof, are also useful as primers and/or templates in a PCRreaction for amplifying genes encoding PTTG proteins.

Antibodies that are immunoreactive with invention PTTG proteins are alsoprovided. These antibodies are useful in diagnostic assays to determinelevels of PTTG proteins present in a given sample, e.g., tissue samples,biological fluids, Western blots, and the like. The antibodies can alsobe used to purify PTTG proteins from crude cell extracts and the like.Moreover, these antibodies are considered therapeutically useful tocounteract or supplement the biological effect of PTTGs in vivo.

Methods and diagnostic systems for determining the levels of PTTGprotein in various tissue samples are provided as well. These diagnosticmethods can be used for monitoring the level of therapeuticallyadministered PTTG protein or fragments thereof to facilitate themaintenance of therapeutically effective amounts. These diagnosticmethods can also be used to diagnose pathologic and physiologicaldisorders that result from abnormal levels of PTTG protein.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the effect of PTTG expression on cell proliferation. Thecell growth rate is expressed as absorbance at 595 nM. The error barsrepresent SEM (n=6). Three independent experiments were performed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention arose from a desire by the inventors to improveover the prior art methods for detection of diseases or conditionsassociated with overproduction of, or abnormalities in, PTTG expression.The inventors set out to isolate a PTTG gene in one species and thenutilized probes based on the thus found gene sequence to probe thegenomic DNA of other species, particularly human, and devise a methodfor mutating the PTTG gene as well as a method for replacing defectivePTTG genes. They, in addition, conceived of a transgenic animal for useas a model for the study of such diseases and conditions in humans. Thisinvention therefore provides isolated, purified mammalianpituitary-tumor-specific-gene (PTTG) proteins, polypeptides, andfragments thereof encoded by the nucleic acid of the invention. As usedherein, the phrase “PTTG” refers to a mammalian family of isolatedand/or substantially pure proteins, preferably human, that are able totransform cells in tissue culture, e.g., NIH 3T3 cells, and the like.The PTTG proteins of the invention have the ability to induce tumorformation in nude mice, e.g., when transfected into NIH 3T3 cells, andthe like. The PTTG proteins of the invention include naturally occurringallelic variants thereof encoded by mRNA generated by alternativesplicing of a primary transcript, and further include fragments whichretain at least one native biological activity, such as immunogenicity.

To identify genes specifically expressed in pituitary tumor cells, theinventors utilized GH-secreting and prolactin-secreting rat pituitarytumor cell lines. In this manner, they utilized them to eliminate theadmixture of normal tissues present in surgically excised humanpituitary tumors and solid experimental rat tumors. Upon screening about30% of expressed MRNA, a pituitary tumor transforming gene (PTTG) wasidentified and characterized. The sequence of PTTG revealed no homologyto any known sequences in the GenBank. The PTTG gene encodes a proteinof 199 amino acids that contains no characterized functional motif,which clearly indicates that PTTG is a novel protein.

The inventors showed the pituitary tumor specific expression of PTTG byNorthern blot analysis. Other than pituitary tumor cells, testis tissueis the only normal non-tumor tissue to show PTTG expression.Interestingly, the PTTG messenger RNA in testis appears to be about 300bp shorter than that of the pituitary tumor, which indicates that thetestis messenger is a PTTG splice variant of the pituitary messenger.

The importance of PTTG in tumorigenesis is illustrated by its ability totransform 3T3 fibroblasts when overexpressed in these cells, as shown bymorphological change and anchorage-independent growth of PTTGtransfectants in soft agar. This finding, moreover, is underscored bythe discovery that multiple tumor cell lines express abundant amounts ofPTTG. Furthermore, nude mice injected with PTTG-expressing 3T3 cellsdeveloped large tumors within 3 weeks at all injection sites. These datashow that PTTG alone is capable of cellular transformation without therequirement of a complimentary oncogene, and that it is potentlytumorigenic in vivo. In general, full cell transformation requires twocomplementary oncogenes. See (Land et al, Nature, 304:696 (1983); Schwabet al., Nature, 316:160 (1985); Ruby et al., Nature 304:602 (1983). Insome cases, however, the overexpression of a single oncogene may besufficient to induce cellular transformation, as is the case in theRat-1 cell transformation by overexpression of the Ras gene alone. See,Reynolds, VL Oncogene, 1:323 (1987). In the present case, the inventorsfound that PTTG does not stimulate cell proliferation in culturedtransfected cells within 72 hours of assaying time. In fact, they foundthat PTTG unexpectedly inhibits all proliferation in culturedtransfected cells. This anti-proliferative effect is similar to thepotent inhibition of cell growth seen by Massague et al. with TGFβ. Oncethe cells are transformed, however, cell proliferation is accelerated,and rapid growth of tumors is seen in nude mice.

The PTTG proteins of this invention are polypeptides selectively boundby anti-PTTG (anti-PTSG) antibody, the antibody preferably binding tothe human protein, including amino acid sequences SEQ ID NO:2, SEQ. IDNo: 4, and fragments 5 to 50 amino acids long which bind to anti-PTTGantibody. The isolated PTTG proteins of the invention are generally freeof other cellular components and/or contaminants normally associatedwith a native in vivo environment, although they may have a certaincontent of these products, such as proteins, RNA, DNA andpolysaccharides.

The PTTG proteins are primarily, although not exclusively, expressed bypituitary tumor cells with expression detected in testis. The transcriptin rat pituitary tumor cells is about 1.3 kb in size while thetranscript in testis is about 1 kb, as observed by a Northern blotassay. Splice variant cDNA transcripts encoding a PTTG family ofproteins are clearly also contemplated by the present invention.

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptide orproteins means that the DNA, RNA, polypeptide or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment. As a result ofthis human intervention, the recombinant DNAs, RNAs, polypeptide andproteins of the invention are useful in ways described herein that theDNAS, RNAS, polypeptide or proteins as they naturally occur are not.

As used herein, “mammalian” refers to the variety of species from whichthe PTTG protein of the invention is derived from, e.g., human, rat,mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline,and the like. A preferred PTTG protein herein, is human PTTG.

Also part of this invention is the PTTG gene, which when defective orpresent, is responsible for pituitary tumorigenesis. A search of GenBankand protein profile analysis (BLAST Program search of databases of thenational center for Biotechnology Information) indicated that PTTGshares no homology with known sequences, and its encoded protein ishighly hydrophilic, and contains no well recognized functional motifs.

Presently preferred PTTG proteins of the invention include amino acidsequences that are substantially the same as the amino acid sequence SEQID NO:2, SEQ. ID No: 4, and fragments thereof about 5 to 50 amino acidslong, as well as biologically active, modified forms thereof. Those ofskill in the art will recognize that numerous residues of theabove-described sequences can be substituted with other, chemically,sterically and/or electronically similar residues without substantiallyaltering the biological activity of the resulting receptor species. Inaddition, larger polypeptide sequences containing substantially the samesequence as SEQ ID NO:2 therein (e.g., splice variants) arecontemplated.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptide (or nucleic acids referred to hereinbefore)containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions, or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

The term “biologically active” or “functional”, when used herein as amodifier of the PTTG protein(s) of this invention or polypeptidefragment thereof, refers to a polypeptide that exhibits at least one ofthe functional characteristics attributed to PTTG. For example, onebiological activity of PTTG is the ability to transform cells in vitro(e.g., NIH 3T3 cells, and the like). Yet another biological activity ofPTTG is the ability to induce tumor formation in nude mice (e.g., whentransfected into NIH 3T3 cells, and the like).

PTTG is also active as an immunogen for the production of polyclonal andmonoclonal antibodies that bind selectively to PTTG. Thus, an inventionnucleic acid encoding PTTG will encode a polypeptide specificallyrecognized by an antibody that also specifically recognizes the PTTGprotein, preferably human, including amino acid sequences SEQ ID NO:2,SEQ. ID No: 4, and fragments thereof about 5 to 50 amino acids longwhich bind to anti-PTTG antibody. Such activity may be assayed by anymethod known to those of skill in the art. For example, atest-polypeptide encoded by a PTTG cDNA may be used to produceantibodies, which are then assayed for their ability to bind to theprotein including the sequence set forth in SEQ ID NO:2. If the antibodybinds to the test-polypeptide and the protein including the sequence setforth in SEQ ID NO:2 with substantially the same affinity, then thepolypeptide possesses the requisite biological activity.

The PTTG proteins of the invention may be isolated by methods well-knownin the art, e.g., the recombinant expression systems described herein,precipitation, gel filtration, ion-exchange, reverse-phase and affinitychromatography, and the like. Other well-known methods are described inDeutscher et al., Guide to Protein Purification: Methods in EnzymologyVol. 182, (Academic Press, (1990)), which is incorporated herein byreference. Alternatively, the isolated polypeptide of the presentinvention can be obtained using well-known recombinant methods asdescribed, for example, in Sambrook et al., supra., 1989).

An example of the means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding the PTTG in a suitable host cell, such asa bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte), or amammalian cell, using methods well known in the art, and recovering theexpressed polypeptide, again using well-known methods. The PTTGpolypeptide of the invention may be isolated directly from cells thathave been transformed with expression vectors as described herein. Theinvention polypeptide, biologically active fragments, and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptide can be produced using Applied Biosystems,Inc. Model 430A or 431A automatic peptide synthesizer (Foster City,Calif.) employing the chemistry provided by the manufacturer.

Also encompassed by the term PTTG are polypeptide fragments orpolypeptide analogs thereof. The term “polypeptide analog” includes anypolypeptide having an amino acid residue sequence substantiallyidentical to a sequence specifically shown herein in which one or moreresidues have been conservatively substituted with a functionallysimilar residue and which displays the ability to mimic PTTG asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another. The phrase “conservativesubstitution” also includes the use of a chemically derivatized residuein place of a non-derivatized residue, provided that such polypeptidedisplays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include, for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptide of the present invention also include any polypeptide havingone or more additions and/or deletions of residues, relative to thesequence of a polypeptide whose sequence is shown herein, so long as therequisite activity is maintained.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified PTTG polypeptide, anactive fragment or polypeptide analog thereof, or a purified, matureprotein and active fragments thereof, alone or in combination with eachother. These polypeptide or proteins can be recombinantly derived,chemically synthesized or purified from native sources. As used herein,the term “acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents.

In accordance with another embodiment of the present invention, thereare provided isolated nucleic acids, which encode the PTTG(pituitary-tumor-transforming-gene) proteins of the invention, andfragments thereof. The nucleic acid molecules described herein areuseful for producing invention proteins, when such nucleic acids areincorporated into a variety of protein expression systems known to thoseof skill in the art. In addition, such nucleic acid molecules orfragments thereof can be labeled with a readily detectable substituentand used as hybridization probes for assaying for the presence and/oramount of a PTTG gene or mRNA transcript in a given sample. The nucleicacid molecules described herein, and fragments thereof, are also usefulas primers and/or templates in a PCR reaction for amplifying genesencoding the invention protein described herein.

The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a PTTG protein. Onemeans of isolating a nucleic acid encoding an PTTG polypeptide is toprobe a mammalian genomic library with a natural or artificiallydesigned DNA probe using methods well known in the art. DNA probesderived from the PTTG gene are particularly useful for this purpose. DNAand cDNA molecules that encode PTTG polypeptide can be used to obtaincomplementary genomic DNA, cDNA or RNA from mammalian (e.g., human,mouse, rat, rabbit, pig, and the like), or other animal sources, or toisolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods described in more detail below. Examplesof nucleic acids are RNA, cDNA, or isolated genomic DNA encoding an PTTGpolypeptide. Such nucleic acids may include, but are not limited to,nucleic acids comprising SEQ ID NO:1, alleles thereof, preferably atleast nucleotides 293-889 of SEQ ID NO:1 or splice variant cDNAsequences thereof.

As used herein, the phrases “splice variant” or “alternatively spliced”,when used to describe a particular nucleotide sequence encoding aninvention receptor, refers to a cDNA sequence that results from the wellknown eukaryotic RNA splicing process. The RNA splicing process involvesthe removal of introns and the joining of exons from eukaryotic primaryRNA transcripts to create mature RNA molecules of the cytoplasm. Methodsof isolating splice variant nucleotide sequences are well known in theart. For example, one of skill in the art may employ nucleotide probesderived from the PTTG encoding DNA of SEQ ID NO:1, SEQ. ID No: 3,alleles thereof, splice variants thereof or fragments thereof about 10to 150 nucleotide long and their anti-sense nucleic acids to screen thecDNA or genomic library of the same or other species as describedherein.

In one embodiment of the present invention, DNAs encoding the PTTGproteins of this invention comprise SEQ. ID NO:1, SEQ. ID No: 3, allelesthereof, splice variants thereof and fragments thereof about 15 to 150nucleotide long and anti-sense nucleic acids thereof. In anotherembodiment of the present invention, DNA molecules encoding theinventive proteins comprise nucleotides 293-889 of SEQ ID NO:1, allelesthereof, splice variants thereof and fragments thereof about 10 to 150nucleotide long. In yet another embodiment, the DNA comprisesnucleotides 95-700 of SEQ ID NO:3, alleles thereof, splice variantsthereof and fragments thereof about 10 to 150 nucleotides long.

As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in SEQ ID NO:2, or a larger amino acid sequenceincluding SEQ ID NO:2. In another embodiment, DNA having “substantiallythe same nucleotide sequence” as the reference nucleotide sequence hasat least 60% identity with respect to the reference nucleotide sequence.DNA having at least 70%, more preferably at least 90%, yet morepreferably at least 95 %, identity to the reference nucleotide sequenceis preferred.

The present invention also encompasses nucleic acids which differ fromthe nucleic acids shown in SEQ ID NO:1, but which have the samephenotype. Phenotypically similar nucleic acids are also referred to as“functionally equivalent nucleic acids”. As used herein, the phrase“functionally equivalent nucleic acids” encompasses nucleic acidscharacterized by slight and non-consequential sequence variations thatwill function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptide that are thesame as those disclosed herein or that have conservative amino acidvariations, or that encode larger polypeptide that include SEQ ID NO:2.For example, conservative variations include substitution of a non-polarresidue with another non-polar residue, or substitution of a chargedresidue with a similarly charged residue. These variations include thoserecognized by skilled artisans as those that do not substantially alterthe tertiary structure of the protein.

Further provided are nucleic acids encoding PTTG polypeptides that, byvirtue of the degeneracy of the genetic code, do not necessarilyhybridize to the invention nucleic acids under specified hybridizationconditions. Preferred nucleic acids encoding the PTTG polypeptide of theinvention comprise nucleotides encoding SEQ ID NO:2, SEQ. ID No: 4, andfragments thereof about 5 to 50 amino acids long. Exemplary nucleicacids encoding a PTTG protein of the invention may be selected from thefollowing.

(a) DNA encoding the amino acid sequence set forth in SEQ. ID No.2,

(b) DNA that hybridizes to the DNA of (a) under moderately stringentconditions, wherein said DNA encodes biologically active PTTG, or

(c) DNA degenerate with respect to either (a) or (b) above, wherein saidDNA encodes biologically active PTTG.

Hybridization refers to the binding of complementary strands of nucleicacid (i.e., sense:antisense strands or probe:target-DNA) to each otherthrough hydrogen bonds, similar to the bonds that naturally occur inchromosomal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% identity, preferably about 75% identity, morepreferably about 85% identity to the target DNA; with greater than about90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018 M NaCl at 65° C. (i.e., if a hybrid is notstable in 0.018 M NaCl at 65° C., it will not be stable under highstringency conditions, as contemplated herein). High stringencyconditions can be provided, for example, by hybridization in 50%formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed bywashing in 0.1×SSPE, and 0.1% SDS at 65° C.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5×Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989) are well known to those of skill in the art as are othersuitable hybridization buffers.

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid, e.g., SEQ ID NO:1 orSEQ. ID No: 3, but encode the same amino acids as the reference nucleicacid. For example, codons specified by the triplets “UCU”, “UCC”, “UCA”,and “UCG” are degenerate with respect to each other since all four ofthese codons encode the amino acid serine.

Preferred nucleic acids encoding the invention polypeptide(s) hybridizeunder moderately stringent, preferably high stringency, conditions tosubstantially the entire sequence, or substantial portions, i.e.,typically at least 15-30 nucleotide, of SEQ ID NO:1, SEQ. ID No: 3,although longer fragments are also contemplated.

Site-directed mutagenesis of any region of PTTG cDNA is contemplatedherein for the production of mutant PTTG cDNAs. For example, theTransformer Mutagenesis Kit (available from Clontech) can be used toconstruct a variety of missense and/or nonsense mutations to PTTG cDNA.

The inventive nucleic acids can be produced by a variety of methodswell-known in the art, e.g., the methods described herein, employing PCRamplification using oligonucleotide primers from various regions of SEQID NO:1, and the like.

In accordance with a further embodiment of the present invention,optionally labeled PTTG-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleic acid sequences encoding related novel mammalian PTTGproteins. Construction of mammalian cDNA and genomic libraries,preferably a human library, is well-known in the art. Screening of sucha cDNA or genomic library is initially carried out under low-stringencyconditions, which comprise a temperature of less than about 42° C., aformamide concentration of less than about 50%, and a moderate to lowsalt concentration.

Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5×standard saline citrate (SSC; 20×SSCcontains 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially thesame, i.e., similar, sequence as the coding region of the nucleic acidsof the invention, preferably as nucleotides 293-889 of SEQ ID NO: 1 areobtained.

As used herein, a nucleic acid “probe” is single-stranded DNA or RNA, oranalogs thereof, that has a sequence of nucleotide that includes atleast 14, preferably at least 20, more preferably at least 50,contiguous bases that are the same as (or the complement of) any 14 ormore contiguous bases set forth in any of SEQ ID NO: 1 or SEQ. ID No: 3.Preferred regions from which to construct probes include 5′ and/or 3′coding regions of SEQ ID NO:1. In addition, the entire cDNA encodingregion of an invention PTTG protein, or the entire sequencecorresponding to SEQ ID NO:1, may be used as a probe. Probes may belabeled by methods well-known in the art, as described hereinafter, andused in various diagnostic kits.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptide, and proteins, is well known in the art.For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984) and U.S. Pat. No. 4,493,795.

Also provided are antisense oligonucleotides having a sequence capableof binding specifically with any portion of an mRNA that encodes PTTGpolypeptide so as to prevent translation of the mRNA. The antisenseoligonucleotide may have a sequence capable of binding specifically withany portion of the sequence of the cDNA encoding PTTG polypeptide. Asused herein, the phrase “binding specifically” encompasses the abilityof a nucleic acid sequence to recognize a complementary nucleic acidsequence and to form double-helical segments therewith via the formationof hydrogen bonds between the complementary base pairs. An example of anantisense oligonucleotide is an antisense oligonucleotide comprisingchemical analogs of nucleotide.

Compositions comprising an amount of the antisense oligonucleotide,described above, effective to reduce expression of PTTG polypeptide bypassing through a cell membrane and binding specifically with mRNAencoding PTTG polypeptide so as to prevent translation and an acceptablehydrophobic carrier capable of passing through a cell membrane are alsoprovided herein. Suitable hydrophobic carriers are described, forexample, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and thelike. The acceptable hydrophobic carrier capable of passing through cellmembranes may also comprise a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type. The structure may be part of a protein known tobind to a cell-type specific receptor.

Antisense oligonucleotide compositions are useful to inhibit translationof mRNA encoding invention polypeptide. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind to mRNAencoding PTTG polypeptide and inhibit translation of mRNA and are usefulas compositions to inhibit expression of PTTG associated genes in atissue sample or in a subject.

In accordance with another embodiment of the invention, kits areprovided for detecting mutations, duplications, deletions,rearrangements or aneuploidies in the PTTG gene comprising at least oneinvention PTTG probe or antisense nucleotide.

The present invention provides means to modulate levels of expression ofPTTG polypeptide by employing synthetic antisense oligonucleotidecompositions (hereinafter SAOC) which inhibit translation of mRNAencoding these polypeptide. Synthetic oligonucleotides, or otherantisense chemical structures designed to recognize and selectively bindto mRNA, are constructed to be complementary to portions of the PTTGcoding strand or nucleotide sequences shown in SEQ. ID No: 1 or SEQ. IDNo: 3. The SAOC is designed to be stable in the blood stream foradministration to a subject by injection or by direct tumor siteintegration, or stable in laboratory cell culture conditions. The SAOCis designed to be capable of passing through the cell membrane in orderto enter the cytoplasm of the cell by virtue of physical and chemicalproperties of the SAOC which render it capable of passing through cellmembranes, for example, by designing small, hydrophobic SAOC chemicalstructures, or by virtue of specific transport systems in the cell whichrecognize and transport the SAOC into the cell. In addition, the SAOCcan be designed for administration only to certain selected cellpopulations by targeting the SAOC to be recognized by specific cellularuptake mechanisms which bind and take up the SAOC only within selectcell populations.

For example, the SAOC may be designed to bind to a receptor found onlyin a certain cell type, as discussed supra. The SAOC is also designed torecognize and selectively bind to target mRNA sequence, which maycorrespond to a sequence contained within the sequence shown in SEQ IDNO:1. The SAOC is designed to inactivate target mRNA sequence by eitherbinding thereto and inducing degradation of the mRNA by, for example,RNase I digestion, or inhibiting translation of mRNA target sequence byinterfering with the binding of translation-regulating factors orribosomes, or inclusion of other chemical structures, such as ribozymesequences or reactive chemical groups which either degrade or chemicallymodify the target mRNA. SAOCs have been shown to be capable of suchproperties when directed against mRNA targets (see Cohen et al., TIPS,10:435 (1989) and Weintraub, Sci. American, January (1990), p.40; bothincorporated herein by reference).

In accordance with yet another embodiment of the present invention,there is provided a method for the recombinant production of the PTTGprotein(s) of the invention by expressing the above-described nucleicacid sequences in suitable host cells. Recombinant DNA expressionsystems that are suitable to produce PTTG proteins described herein arewell-known in the art. For example, the above-described nucleotidesequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof.

Suitable expression vectors are well-known in the art, and includevectors capable of expressing DNA operatively linked to a regulatorysequence, such as a promoter region that is capable of regulatingexpression of such DNA. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. In addition, vectors may containappropriate packaging signals that enable the vector to be packaged by anumber of viral virions, e.g., retroviruses, herpes viruses,adenoviruses, resulting in the formation of a “viral vector”.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector nucleotide sequences,such as promoters, enhancers, transcriptional and translational stopsites, and other signal sequences. For example, operative linkage of DNAto a promoter refers to the physical and functional relationship betweenthe DNA and the promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptide, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and includepBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, Calif.),and the like. Other suitable vectors and promoters are disclosed indetail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989, the disclosureof which is incorporated herein by reference in its entirety.

Other suitable vectors for transformation of E. coli cells include thepET expression vectors (Novagen, see U.S. Pat. No. 4,952,496), e.g.,pET11a, which contains the T7 promoter, T7 terminator, the inducible E.coli lac operator, and the lac repressor gene; and pET 12a-c, whichcontain the T7 promoter, T7 terminator, and the E. coli ompT secretionsignal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al.,Meth. in Enzymology, 153:492-507, 1987), which contains the lpppromoter, the lacUV5 promoter operator, the ompA secretion signal, andthe lac repressor gene.

Exemplary, eukaryotic transformation vectors, include the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system (described byMulligan and Berg, 1979, Nature Vol. 277:108-114) the Okayama-Bergcloning system (Mol. Cell Biol. Vol. 2:161-170, 1982), and theexpression cloning vector described by Genetics Institute (Science Vol.228:810-815, 1985), are available which provide substantial assurance ofat least some expression of the protein of interest in the transformedeukaryotic cell line.

Particularly preferred base vectors which contain regulatory elementsthat can be linked to the invention PTTG-encoding DNAs for transfectionof mammalian cells are cytomegalovirus (CMV) promoter-based vectors suchas pcDNA1 (Invitrogen, San Diego, Calif.), MMTV promoter-based vectorssuch as pMAMNeo (Clontech, Palo Alto, Calif.) and pMSG (Pharmacia,Piscataway, N.J.), and SV40 promoter-based vectors such as pSVβ(Clontech, Palo Alto, Calif.).

In accordance with another embodiment of the present invention, thereare provided “recombinant cells” containing the nucleic acid molecules(i.e., DNA or mRNA) of the present invention. Methods of transformingsuitable host cells, preferably bacterial cells, and more preferably E.coli cells, as well as methods applicable for culturing said cellscontaining a gene encoding a heterologous protein, are generally knownin the art. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (1989).

Exemplary methods of introducing (transducing) expression vectorscontaining invention nucleic acids into host cells to produce transducedrecombinant cells (i.e., cells containing recombinant heterologousnucleic acid) are well-known in the art (see, for review, Friedmann,1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, eachof which are incorporated herein by reference in their entirety).Exemplary methods of transduction include, e.g., infection employingviral vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764),calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and 4,634,665),dextran sulfate transfection, electroporation, lipofection (see, e.g.,U.S. Pat. Nos. 4,394,448 and 4,619,794), cytofection, particle beadbombardment, and the like. The heterologous nucleic acid can optionallyinclude sequences which allow for its extrachromosomal (i.e., episomal)maintenance, or the heterologous DNA can be caused to integrate into thegenome of the host (as an alternative means to ensure stable maintenancein the host).

Host organisms contemplated for use in the practice of the presentinvention include those organisms in which recombinant production ofheterologous proteins has been carried out. Examples of such hostorganisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris;see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk cells), insectcells, and the like. Presently preferred host organisms are bacteria.The most preferred bacteria is E. coli.

In one embodiment, nucleic acids encoding the PTTG proteins of theinvention may be delivered into mammalian cells, either in vivo or invitro using suitable viral vectors well-known in the art, e.g.,retroviral vectors, adenovirus vectors, and the like. In addition, whereit is desirable to limit or reduce the in vivo expression of the PTTG ofthis invention, the introduction of the antisense strand of theinvention nucleic acid is contemplated.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing PTTG nucleic acidencoding a PTTG protein into mammalian cells (e.g., vascular tissuesegments) are well known in the art. These viral vectors include, forexample, Herpes simplex virus vectors (e.g., Geller et al., 1988,Science, 241:1667-1669), Vaccinia virus vectors (e.g., Piccini et al.,1987, Meth. in Enzymology, 153:545-563; Cytomegalovirus vectors(Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.78-84), Moloney murine leukemia virus vectors (Danos et al., 1980, PNAS,USA, 85:6469), adenovirus vectors (e.g., Logan et al., 1984, PNAS, USA,81:3655-3659; Jones et al., 1979, Cell, 17:683-689; Berkner, 1988,Biotechniques, 6:616-626; Cotten et al., 1992, PNAS, USA, 89:6094-6098;Graham et al., 1991, Meth. Mol. Biol., 7:109-127), adeno-associatedvirus vectors, retrovirus vectors, and the like. See, e.g., U.S. Pat.Nos. 4,405,712 and 4,650,764. Especially preferred viral vectors are theadenovirus and retroviral vectors.

For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., 1992, PNAS, USA, 89:6099-6103; Curiel et al., 1992, Hum.Gene Therapy, 3:147-154; Gao et al., 1993, Hum. Gene Ther., 4:14-24) areemployed to transduce mammalian cells with heterologous PTTG nucleicacid. Any of the plasmid expression vectors described herein may beemployed in a TfAdpl-DNA complex.

As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764).

Suitable retroviral vectors for use herein are described, for example,in U.S. Pat. No. 5,252,479, and in WIPO publications WO 92/07573, WO90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated hereinby reference, which provide a description of methods for efficientlyintroducing nucleic acids into human cells using such retroviralvectors. Other retroviral vectors include, for example, the mousemammary tumor virus vectors (e.g., Shackleford et al., 1988, PNAS, USA,85:9655-9659), and the like.

In accordance with yet another embodiment of the present invention,there are provided anti-PTTG antibodies having specific reactivity withPTTG polypeptide of the present invention. Active fragments ofantibodies are encompassed within the definition of “antibody”.Invention antibodies can be produced by methods known in the art usingthe PTTG polypeptide of the invention, proteins or portions thereof asantigens. For example, polyclonal and monoclonal antibodies can beproduced by methods well known in the art, as described, for example, inHarlow and Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory (1988)), which is incorporated herein by reference. The PTSGpolypeptide of the invention may be utilized as immunogens in generatingsuch antibodies. Alternatively, synthetic peptides can be prepared(using commercially available synthesizers) and used as immunogens.Amino acid sequences can be analyzed by methods well known in the art todetermine whether they encode hydrophobic or hydrophilic domains of thecorresponding polypeptide. Altered antibodies such as chimeric,humanized, CDR-grafted or bifunctional antibodies can also be producedby methods well known in the art. Such antibodies can also be producedby hybridoma, chemical synthesis or recombinant methods described, forexample, in Sambrook et al., supra., and Harlow and Lane, supra. Bothanti-peptide and anti-fusion protein antibodies can be used. (see, forexample, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991); Ausubelet al., Current Protocols in Molecular Biology (John Wiley and Sons, NY(1989) which are incorporated herein by reference).

Antibody so produced can be used, inter alia, in diagnostic methods andsystems to detect the level of PTTG protein present in a mammalian,preferably human, body sample, such as tissue or vascular fluid. Suchantibodies can also be used for the immunoaffinity or affinitychromatography purification of the PTTG protein of the invention. Inaddition, methods are contemplated herein for detecting the presence ofPTTG polypeptide either on the surface of a cell or within a cell (suchas within the nucleus), which methods comprise contacting the cell withan antibody that specifically binds to PTTG polypeptide, underconditions permitting binding of the antibody to PTTG polypeptide,detecting the presence of the antibody bound to PTTG, and therebydetecting the presence of invention polypeptide on the surface of, orwithin, the cell. With respect to the detection of such polypeptide, theantibodies can be used for in vitro diagnostic or in vivo imagingmethods.

Immunological procedures useful for in vitro detection of target PTTGpolypeptide in a sample include immunoassays that employ a detectableantibody. Such immunoassays include, for example, ELISA, Pandexmicrofluorimetric assay, agglutination assays, flow cytometry, serumdiagnostic assays and immunohistochemical staining procedures which arewell known in the art. An antibody can be made detectable by variousmeans well known in the art. For example, a detectable marker can bedirectly or indirectly attached to the antibody. Useful markers include,for example, radionuclides, enzymes, fluorogens, chromogens andchemiluminescent labels.

The anti-PTTG antibodies of the invention modulate activity of the PTTGpolypeptide in living animals, in humans, or in biological tissues orfluids isolated therefrom. Accordingly, compositions comprising acarrier and an amount of an antibody having specificity for PTTGpolypeptide effective to block naturally occurring ligands or otherPTTG-binding proteins from binding to invention PTTG polypeptide arecontemplated herein. For example, a monoclonal antibody directed to anepitope of PTTG polypeptide molecules present on the surface of a celland having an amino acid sequence substantially the same as an aminoacid sequence for a cell surface epitope of an PTTG polypeptideincluding the amino acid sequence shown in SEQ ID NO:2, SEQ. ID No: 4,and fragments thereof, may be useful for this purpose.

The present invention further provides transgenic non-human mammals thatare capable of expressing exogenous nucleic acids encoding PTTGpolypeptide. As employed herein, the phrase “exogenous nucleic acid”refers to nucleic acid sequence which is not native to the host, orwhich is present in the host in other than its native environment (e.g.,as part of a genetically engineered DNA construct). In addition tonaturally occurring levels of PTTG, the PTTG proteins of this inventionmay either be overexpressed, underexpressed, or expressed in an inactivemutated form, such as in the well-known knock-out transgenics, intransgenic mammals.

Also provided are transgenic non-human mammals capable of expressingnucleic acids encoding PTTG polypeptide so mutated as to be incapable ofnormal activity, i.e., do not express native PTTG. The present inventionalso provides transgenic non-human mammals having a genome comprisingantisense nucleic acids complementary to nucleic acids encoding PTTGpolypeptide, placed so as to be transcribed into antisense mRNAcomplementary to mRNA encoding PFTG polypeptide, which hybridizes to themRNA and, thereby, reduces the translation thereof. The nucleic acid mayadditionally comprise an inducible promoter and/or tissue specificregulatory elements, so that expression can be induced, or restricted tospecific cell types. Examples of nucleic acids are DNA or cDNA having acoding sequence substantially the same as the coding sequence shown inSEQ ID NO:1. An example of a non-human transgenic mammal is a transgenicmouse.

Animal model systems which elucidate the physiological and behavioralroles of PTTG polypeptide are also provided, and are produced bycreating transgenic animals in which the expression of the PTTGpolypeptide is altered using a variety of techniques. Examples of suchtechniques include the insertion of normal or mutant versions of nucleicacids encoding an PTTG polypeptide by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal. (See, forexample, Hogan et al., Manipulating the Mouse Embryo: A LaboratoryManual (Cold Spring Harbor Laboratory, (1986)).

Also contemplated herein, is the use of homologous recombination ofmutant or normal versions of PTTG genes with the native gene locus intransgenic animals, to alter the regulation of expression or thestructure of PTTG polypeptide (see, Capecchi et al., Science 244:1288(1989); Zimmer et al., Nature 338:150 (1989); which are incorporatedherein by reference). Homologous recombination techniques are well knownin the art. Homologous recombination replaces the native (endogenous)gene with a recombinant or mutated gene to produce an animal that cannotexpress native (endogenous) protein but can express, for example, amutated protein which results in altered expression of PTTG polypeptide.

In contrast to homologous recombination, microinjection adds genes tothe host genome, without removing host genes. Microinjection can producea transgenic animal that is capable of expressing both endogenous andexogenous PTTG protein. Inducible promoters can be linked to the codingregion of nucleic acids to provide a means to regulate expression of thetransgene. Tissue specific regulatory elements can be linked to thecoding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of specific ligands, i.e., agonists andantagonists, which activate or inhibit protein responses.

Invention nucleic acids, oligonucleotides (including antisense), vectorscontaining same, transformed host cells, polypeptide and combinationsthereof, as well as antibodies of the present invention, can be used toscreen compounds in vitro to determine whether a compound functions as apotential agonist or antagonist to the PTTG polypeptide of theinvention. These in vitro screening assays provide information regardingthe function and activity of the PTTG polypeptide of the invention,which can lead to the identification and design of compounds that arecapable of specific interaction with one or more types of polypeptide,peptides or proteins.

In accordance with still another embodiment of the present invention,there is provided a method for identifying compounds which bind to PTTGpolypeptide. The invention proteins may be employed in a competitivebinding assay. Such an assay can accommodate the rapid screening of alarge number of compounds to determine which compounds, if any, arecapable of binding to PTTG proteins. Subsequently, more detailed assayscan be carried out with those compounds found to bind, to furtherdetermine whether such compounds act as modulators, agonists orantagonists of invention proteins.

In another embodiment of the invention, there is provided a bioassay foridentifying compounds which modulate the activity of the PTTGpolypeptide of the invention. According to this method, the PTTGpolypeptides of the invention are contacted with an “unknown” or testsubstance (in the presence of a reporter gene construct when antagonistactivity is tested), the activity of the polypeptide is monitoredsubsequent to the contact with the “unknown” or test substance, andthose substances which cause the reporter gene construct to be expressedare identified as functional ligands for PTTG polypeptide.

In accordance with another embodiment of the present invention,transformed host cells that recombinantly express the PTSG polypeptideof the invention may be contacted with a test compound, and themodulating effect(s) thereof can then be evaluated by comparing thePTTG-mediated response (e.g., via reporter gene expression) in thepresence and absence of test compound, or by comparing the response oftest cells or control cells (i.e., cells that do not express PTTGpolypeptide), to the presence of the compound.

As used herein, a compound or a signal that “modulates the activity” ofPTTG polypeptide of this invention refers to a compound or a signal thatalters the activity of PTTG polypeptide so that the activity of theinvention PTTG polypeptide is different in the presence of the compoundor signal than in the absence of the compound or signal. In particular,such compounds or signals include agonists and antagonists. An agonistencompasses a compound or a signal that activates PTTG protein function.Alternatively, an antagonist includes a compound or signal thatinterferes with PTTG protein function. Typically, the effect of anantagonist is observed as a blocking of agonist-induced proteinactivation. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for agonist binding. A non-competitiveantagonist or blocker inactivates the function of the polypeptide byinteracting with a site other than the agonist interaction site.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate PTTG activity generally requirecomparison to a control. One type of a “control” is a cell or culturethat is treated substantially the same as the test cell or test cultureexposed to the compound, with the distinction that the “control” cell orculture is not exposed to the compound. For example, in methods that usevoltage clamp electrophysiological procedures, the same cell can betested in the presence or absence of compound, by merely changing theexternal solution bathing the cell. Another type of “control” cell orculture may be a cell or culture that is identical to the transfectedcells, with the exception that the “control” cell or culture do notexpress native proteins. Accordingly, the response of the transfectedcell to compound is compared to the response (or lack thereof) of the“control” cell or culture to the same compound under the same reactionconditions.

In yet another embodiment of the present invention, the activation ofPTTG polypeptide can be modulated by contacting the polypeptide with aneffective amount of at least one compound identified by theabove-described bioassays.

In accordance with another embodiment of the present invention, thereare provided methods for diagnosing or detecting a pathological mass(such as, for example, an endocrine or non-endocrine tumor,atherosclerotic plaque, and the like), said method comprising detecting,in cells of a subject, a transcribed or mutant sequence including SEQ IDNO:1.

In a particular embodiment, the invention diagnostic methods describedherein are useful for differential diagnosis of malignant versus benigntumors in biopsy specimens, and the like. In another embodiment, theinvention diagnostic methods described herein are useful for predictingtumor behavior and responsiveness to therapy.

In accordance with another embodiment of the present invention, thereare provided methods for diagnosing pituitary tumors, said methodcomprising detecting, in pituitary-derived cells of a subject, atranscribed mRNA sequence including SEQ ID NO:1.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention nucleic acid in a suitable packaging material. Thediagnostic nucleic acids are derived from the PTTG-encoding nucleicacids described herein. In one embodiment, for example, the diagnosticnucleic acids are derived from SEQ ID NO:1. Invention diagnostic systemsare useful for assaying for the presence or absence of nucleic acidencoding PTTG in either genomic DNA or in transcribed nucleic acid (suchas mRNA or cDNA) encoding PTTG in any tumors (e.g., pituitary, and thelike) or diseased tissue. Invention diagnostic systems contemplatedherein, may make use of well-known polymerase chain reaction (PCR) orRTPCR (reverse-transcriptase-PCR) methodologies.

A suitable diagnostic system includes at least one invention nucleicacid, preferably two or more invention nucleic acids, as a separatelypackaged chemical reagent(s) in an amount sufficient for at least oneassay. Instructions for use of the packaged reagent are also typicallyincluded. Those of skill in the art can readily incorporate inventionnucleic probes and/or primers into kit form in combination withappropriate buffers and solutions for the practice of the inventionmethods as described herein.

As employed herein, the phrase “packaging material” refers to one ormore physical structures used to house the contents of the kit, such asinvention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular sequence encoding PTTG including the nucleotidesequence set forth in SEQ ID NO:1 or a mutant thereof, therebydiagnosing the presence of, or a predisposition for, a particularpathology (such as, for example, pituitary tumorigenesis, and the like).In addition, the packaging material contains instructions indicating howthe materials within the kit are employed both to detect a particularsequence and diagnose the presence of, or a predisposition for, aparticular pathology.

The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

All US patents and all publications mentioned herein are incorporated intheir entirety by reference thereto. The invention will now be describedin greater detail by reference to the following non-limiting examples.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); or Methods in Enzymology: Guide to Molecular Cloning TechniquesVol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987).

EXAMPLES Example 1

Isolation of PTTG cDNA

To clarify the molecular mechanisms involved in pituitary tumorigenesis,differential display PCR was used to identify mRNAs differentiallyexpressed in pituitary tumor cells (see, e.g., Risinger et al., 1994,Molec. Carcinogenesis, 11:13-18; and Qu et al., 1996, Nature,380:243-247). GC and GH₄ pituitary tumor cell lines (ATCC #CCL-82 and#CCL-82.1, respectively) and an osteogenic sarcoma cell line UM108 (ATCC#CRL-1663) were grown in DMEM supplemented with 10% fetal bovine serum.Normal Sprague-Dawley rat pituitaries were freshly excised. Total RNAwas extracted from tissue cultured cells and pituitary tissue usingRNeasy™ kit (Qiagen) according to manufacturer's instructions. Trace DNAcontamination in RNA preparations was removed by DNase1 (GenHunterCorporation) digestion. cDNA was synthesized from 200 ng total RNA usingMMLV reverse transcriptase (GenHunter Corporation), and one of the threeanchored primers (GenHunter Corporation). The cDNA generated was used inthe PCR display.

Three downstream anchored primers AAGCT₁₁N (where “N” may be A, G, or C;SEQ ID NO:7), were used in conjunction with 40 upstream arbitraryprimers for PCR display. 120 primer pairs were used to screen mRNAexpression in pituitary tumors versus normal pituitary. One tenth of thecDNA generated from the reverse transcriptase reaction was amplifiedusing AmpliTaq DNA polymerase (Perkin Elmer) in a total volume of 20 μlcontaining 10 mM Tris, pH 8.4, 50 nM KCl, 1.5 mM MgCl₂, 0.001% gelatin,2 μM dNTPs, 0.2 μM each primer and 1 μl [³⁵S]dATP. PCR cycles consistedof 30 seconds at 94° C., 2 minutes at 40° C., and 30 seconds at 72° C.for 40 cycles. The products were separated on 6% sequencing gels, anddried gels were exposed to Kodak film for 24 to 48 hours.

After development, DNA fragments amplified from pituitary tumor andnormal pituitary were compared. Bands unique to pituitary tumor wereexcised from the gel, and DNA extracted by boiling in 100 μl water andprecipitated with ethanol in the presence of glycogen (GenHunterCorporation). DNA was reamplified using the original set of primers andthe same thermal cycling conditions except that the dNTP concentrationwas increased to 20 μM. Reaction products were run on 1% agarose gel andstained with ethidium bromide. Bands were excised from the gel, eluted(Qiagen), cloned in to TA vectors (Invitrogen) and sequenced usingsequenase (USB). Using 120 primer pairs in the above-described PCRassay, 11 DNA bands that appeared to be differentially expressed inpituitary tumor cells were identified. These bands were evaluatedfurther by Northern blot analysis, using the PCR products as probes.

For Northern blot analysis, 20 μg of total RNA were fractionated on 1%agarose gel, blotted on to nylon membrane and hybridized with randomprimed probe using Quickhyb solutions (Stratagene). After washing,membranes were exposed to Kodak films for 6 to 72 hours. As a result ofthe Northern blot assay, pituitary tumor specific signals were detectedfor 2 bands. DNA sequence analysis revealed that one sequence washomologous with Insulin-induced growth response protein, while theanother 396 base pair fragment (amplified using 5′ AAGCTTTTTTTTTTTG 3′[SEQ ID NO:5] as the anchored primer and 5′ AAGCTTGCTGCTC 3′ [SEQ IDNO:6] as an arbitrary primer) showed no homology to known sequences inthe GenBank. This 396 bp fragment detected a highly expressed mRNA ofabout 1.3 kb in pituitary tumor cells, but not in normal pituitary norin osteogenic sarcoma cells.

Example 2

Characterization of PTTG cDNA

To characterize this pituitary tumor-specific mRNA further, a cDNAlibrary was constructed using mRNA isolated from rat pituitary tumorcells. Poly A+RNA was isolated from pituitary tumor GH₄ cells usingmessenger RNA isolation kit (Stratagene) according to manufacturer'sinstructions, and was used to construct a cDNA library in ZAP Expressvectors (Stratagene). The cDNA library was constructed using ZAPExpress™ cDNA synthesis and Gigapack III gold cloning kit (Stratagene)following manufacturer's instructions. The library was screened usingthe 396 bp differentially displayed PCR product (cloned into TA vector)as the probe. After tertiary screening, positive clones were excised byin vivo excision using helper phage. The resulting pBK-CMV phagemidcontaining the insert was identified by Southern Blotting analysis.Unidirectional nested deletions were made into the DNA insert usingEXOIII/Mung bean nuclease deletion kit (Stratagene) followingmanufacturer's instructions. Both strands of the insert DNA weresequenced using Sequenase (USB).

Using the 396 bp PCR fragment described in Example 1 as a probe, a cDNAclone of 974 bp was isolated and characterized. This cDNA was designatedas pituitary-tumor-transforming gene (PTTG). The sequence of PTTGcontains an open reading frame for 199 amino acids (SEQ ID NO:2). Thepresence of an in-frame stop codon upstream of the predicted initiationcodon indicates that PTTG contains the complete ORF. The nucleic acidand protein sequences are provided in Table 1 below.

TABLE 1 PTTG Nucleic Acid and Protein Sequences AATTCGGCAC GAGCCAACCTTGAGCATCTG ATCCTCTTGG CTTCTCCTTC CTATCGCTGA  60 GCTGGTAGGC TGGAGACAGTTGTTTGGGTG CCAACATCAA CAAACGATTT CTGTAGTTTA 120 GCGTTTATGA CCCTGGCGTGAAGATTTAAG GTCTGGATTA AGCCTGTTGA CTTCTCCAGC 180 TACTTCTAAA TTTTTGTGCATAGGTGCTCT GGTCTCTGTT GCTGCTTAGT TCTTCCAGCC 240 TTCCTCAATG CCAGTTTTATAATATGCAGG TCTCTCCCCT CAGTAATCCA GG ATG 295                                                          Met                                                            1 GCT ACTCTG ATC TTT GTT GAT AAG GAT AAC GAA GAG CCA GGC AGC CGT 343 Ala Thr LeuIle Phe Val Asp Lys Asp Asn Glu Glu Pro Gly Ser Arg              5                  10                  15 TTG GCA TCT AAGGAT GGA TTG AAG CTG GGC TCT GGT GTC AAA GCC TTA 391 Leu Ala Ser Lys AspGly Leu Lys Leu Gly Ser Gly Val Lys Ala Leu         20                  25                  30 GAT GGG AAA TTG CAGGTT TCA ACG CCA CGA GTC GGC AAA GTG TTC GGT 439 Asp Gly Lys Leu Gln ValSer Thr Pro Arg Val Gly Lys VaL Phe Gly     35                  40                  45 GCC CCA GGC TTG CCT AAAGCC AGC AGG AAG GCT CTG GGA ACT GTC AAC 487 Ala Pro Gly Leu Pro Lys AlaSer Arg Lys Ala Leu Gly Thr Val Asn 50                  55                  60                  65 AGA CTTACT GAA AAG CCA GTG AAG AGT AGT AAA CCC CTG CAA TCG AAA 535 Arg Val ThrGlu Lys Pro Val Lys Ser Ser Lys Pro Leu Gln Ser Lys                 70                  75                  80 CAG CCG ACTCTG AGT CTG AAA AAG ATC ACC GAG AAG TCT ACT AAG ACA 583 Gln Pro Thr LeuSer Val Lys Lys Ile Thr Glu Lys Ser Thr Lys Thr             85                  90                  95 CAA GGC TCT GCTCCT GCT CCT GAT GAT GCC TAC CCA GAA ATA CAA AAG 631 Gln Gly Ser Ala ProAla Pro Asp Asp Ala Tyr Pro Glu Ile Glu Lys        100                 105                 110 TTC TTC CCC TTC GATCCT CTA GAT TTT GAG AGT TTT GAC CTG CCT GAA 679 Phe Phe Pro Phe Asp ProLeu Asp Phe Glu Ser Phe Asp Leu Pro Glu    115                 120                 125 GAG CAC CAG ATC TCA CTTCTC CCC TTG AAT GGA GTG CCT CTC ATG ATC 727 Glu His Gln Ile Ser Leu LeuPro Leu Asn Gly Val Pro Leu Met Ile130                 135                 140                 145 CTG AATGAA GAG AGG GGG CTT GAG AAG CTG CTG CAC CTG GAC CCC CCT 775 Leu Asn CluGlu Arg Gly Leu Glu Lys Leu Leu His Leu Asp Pro Pro                150                 155                 160 TCC CCT CTGCAG AAG CCC TTC CTA CCC TGG GAA TCT GAT CCG TTG CCG 823 Ser Pro Leu GlnLys Pro Phe Leu Pro Trp Glu Ser Asp Pro Leu Pro            165                 170                 175 TCT CCT CCC AGCGCC CTC TCC GCT CTG GAT GTT GAA TTG CCG CCT GTT 871 Ser Pro Pro Ser AlaLeu Ser Ala Leu Asp Val Glu Leu Pra Pro Val        180                 185                 190 TGT TAC GAT GCA GATATT TAAACGTCTT ACTCCTTTAT AGTTTATGTA 919 Cys Tyr Asp Ala Asp Ile (SEQ IDNO:2)     195                 200 AGTTGTATTA ATAAAGCATT TGTGTGTAAAAAAAAAAAAA AAAACTCGAG AGTAC 974 (SEQ ID NO:1)

This was verified by demonstrating both in vitro transcription and invitro translation of the gene product as described in Example 3.

Example 3

In vitro Transcription & Translation of PTTG

Sense and antisense PTTG mRNAs were in vitro transcribed using T3 and T7RNA polymerase (Stratagene), respectively. The excess template wasremoved by DNase I digestion. The in vitro transcribed mRNA wastranslated in rabbit reticular lysate (Stratagene). Reactions werecarried out at 30° C. for 60 minutes, in a total volume of 25 μlcontaining 3 μl in vitro transcribed RNA, 2 μl ³⁵S-Methionine (Dupond)and 20 μl lysate. Translation products were analyzed by SDS-PAGE (15%resolving gel and 5% stacking gel), and exposed to Kodak film for 16hours.

The results indicate that translation of in vitro transcribed PTTG sensemRNA results in a protein of approximately 25 KD on SDS-PAGE, whereas noprotein was generated in either the reaction without added mRNA or whenPTTG antisense mRNA was utilized.

Example 4

Expression of PTTG mRNA

A search of GenBank and a protein profile analysis (using a BLASTProgram search of databases of the national center for BiotechnologyInformation) indicated that PTTG shares no homology with knownsequences, and its encoded protein is highly hydrophilic, and containsno well recognized functional motifs. The tissue expression patten ofPTTG mRNA was studied by Northern Blot analysis. A rat multiple tissueNorthern blot was purchased from Clontech. Approximately 2 μg of polyA+RNA per lane from eight different rat tissues (heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis) was run on adenaturing formaldehyde 1.2% agarose gel, transferred to nylon membraneand UV-cross linked. The membrane was first hybridized to the fulllength PTTG cDNA probe, and was stripped and rehybridized to a humanβ-actin cDNA control probe. Hybridization was performed at 60° C. forone hour in ExpressHyb hybridization solution (Clontech). Washing wastwice 15 minutes at room temperature in 2×SSC, 0.05%SDS, and twice 15minutes at 50° C. in 0.1%SSC, 0.1%SDS. Exposure time for PTTG probe was24 hrs, and actin probe 2 hours.

The results of the Northern assay indicate that testis is the onlytissue, other than pituitary tumor cells, that expresses PTTG mRNA, andthe testis expression level is much lower (2 μg polyA+mRNA, 24 hourexposure) than in pituitary tumor cells (20 μg total RNA, 6 hourexposure). Interestingly, the testicular transcript (about 1 Kb) isshorter than the transcript in pituitary tumors (1.3 Kb), indicatingthat the mRNA is differentially spliced in testis, and that the 1.3 Kbtranscript is specific for pituitary tumor cells.

Example 5

Over-expression of PTTG by NIH 3T3 Fibroblast Cells

Since PTTG mRNA is over-expressed in pituitary tumor cells, whether thisprotein exerts an effect on cell proliferation and transformation wasdetermined. An eukaryotic expression vector containing the entire codingregion of PTTG was stably transfected into NIH 3T3 fibroblasts.

The entire coding region of the PTTG was cloned in frame into pBK-CMVeukaryotic expression vector (Stratagene), and transfected into NIH 3T3cells by calcium precipitation. 48 hrs after transfection, cells werediluted 1:10 and grown in selection medium containing 1 mg/ml G418 fortwo weeks in when individual clones were isolated. Cell extracts wereprepared from each colony and separated on 15% SDS-polyacrylamide gels,and blotted onto nylon membrane. A polyclonal antibody was generatedusing the first 17 amino acids of PTTG as epitope (Research Genetics).The antibody was diluted 1:5000 and incubated with the above membrane atroom temperature for 1 hour. After washing, the membrane was incubatedwith horseradish peroxidase-labeled secondary antibody for one hour atroom temperature. The hybridization signal was detected by enhancedchemiluminescence (ECL detection system, Amersham).

Expression levels of the PTTG were monitored by immunoblot analysisusing the above-described specific polyclonal antibody directed againstthe first 17 amino acids of the protein. Expression levels of individualclones varied, and clones that expressed higher protein levels were usedfor further analysis.

Example 6

Effect of PTTG Expression on Cell Proliferation

A nonradioactive cell proliferation assay was used to determine theeffect of PTTG protein over-expression on cell proliferation (see, e.g.,Mosmann, T., 1983, J. Immunol. Meth., 65:55-63; and Carmichael et al.,1987, Cancer Res., 47:943-946). Cell proliferation was assayed usingCellTiter 96TM Non-radioactive cell proliferation Assay kit (Promega)according to the manufacturer's instructions. Five thousand cells wereseeded in 96 well plates (6 wells for each clone in each assay), andincubated at 37° C. for 24 to 72 hours. At each time point, 15 μl of theDye solution were added to each well, and incubated at 37° C. for 4hours. One hundred μl of the solubilization/stop solution were thenadded to each well. After one hour incubation, the contents of the wellswere mixed, and absorbance at 595 nm was recorded using an ELISA reader.Absorbance at 595 nm correlates directly with the number of cells ineach well.

Three independent experiments were performed and the results are shownin FIG. 1. In FIG. 1, the cell growth rate is expressed as absorbance at595 nM. The error bars represent SEM (n=6). The results (FIG. 1) showthat the growth rate of 3T3 cells expressing PTTG protein (assayed bycellular conversion of tetrazolium into formazan) was suppressed 25 to50% as compared with 3T3 cells expressing the pCMV vector alone,indicating that PTTG protein inhibits cell proliferation.

Example 7

PTTG Induction of Morphological Transformation and Soft-agar Growth ofNIH 3T3 Cells

The transforming property of PTTG protein was demonstrated by itsability to form foci in monolayer cultures and showanchorage-independent growth in soft agar, as shown in Table 2 below.

TABLE 2 Colony Formation by NIH 3T3 Cells Transfected with PTTG cDNAConstructs Efficiency of Colony Cell line Growth in Soft Agar formationin Soft Agar (%) No DNA 0 0 Vector only 1.3 ± 0.7 0.013 PTTG 3  26 ± 4.60.26 PTTG 4 132 ± 26  1.32 PTTG 8  33 ± 6.0 0.33 PTTG 9 72 ± 13 0.72PTTG 10 92 ± 18 0.92 10⁴ cells/dish were plated as described in FIG. 7.Plates were scored at 14 days. Only colonies consist of at least 40cells were counted. Values are means ± SEM of triplicates. Efficiency ofcolony formation was calculated as percentage of number of coloniesdivided by total number of cells.

As primary pituitary cells are an admixture of multiple cell types andthey do not replicate in vitro, NIH 3T3 cells were employed. For thesoft agar assay, 60 mm tissue culture plates were coated with 5 mlsoft-agar (20% 2×DMEM, 50% DMEM, 10% fetal bovine serum, 20% 2.5% agar,melted and combined at 45° C.). See, Schwab et al., 1985, Nature,316:160-162. 2 ml cells suspended in medium were then combined with 4 mlagar mixture, and 1.5 ml of this mixture added to each plate. The cellswere plated at a density of 10⁴ cells/dish and incubated for 14 daysbefore counting the number of colonies and photography.

The results indicate that NIH 3T3 parental cells and 3T3 cellstransfected with pCMV vector do not form colonies on soft agar, whereas3T3 cells transfected with PTTG form large colonies. In addition, focaltransformation is observed in cells over-expressing PTTG protein, butcells expressing pCMV vector without the PTTG insert showed similarmorphology to the parental 3T3 cells.

Example 8

Determination of In vivo PTTG Tumorigenicity

To determine whether PTTG is tumorigenic in vivo, PTTG-transfected 3T3cells were injected subcutaneously into athymic nude mice. 3×10⁵ cellsof either PTTG or pCMV vector alone transfected cells were resuspendedin PBS and injected subcutaneously into nude mice (5 for each group).Tumors were excised from animals at the end of the 3rd week andweighted. All injected animals developed large tumors (1-3 grams) within3 weeks. The results are shown in Table 3 below. No mouse injected withvector only transfected cells developed tumors.

TABLE 3 In vivo Tumorigenesis by NIH 3T3 Cells Transfected with PTTGcDNA Expression Vector Cell line Animals injected Tumor formation Vectoronly 5 0/5 PTTG 4 5 5/5 3 × 10⁵ cells were injected subcutaneously intoathymic nude mice. Tumors developed within 3 weeks and weighed 1-3 gramsat sacrifice.

These results clearly indicate that PTTG is a potent transforming genein vivo.

Example 9

Human Carcinoma Cell Lines Express PTTG

The expression of PTTG in various human cell lines was studied employinga multiple human cancer cell line Northern blot (Clontech). The specificcell lines tested are shown in Table 4 below.

TABLE 4 Human Carcinoma Cell Lines Tested Cell Line PTTG Expression 1Promyelocytic Leukemia HL-60 + 2 HeLa Cell S3 + 3 Chronic MyelogenousLeukemia K-562 + 4 Lymphoblastic Leukemia MOLT-4 + 5 Burkitt's lymphomaRaji + 6 Colorectal Adenocarcinoma SW 480 + 7 Lung Carcinoma A549 + 8Melanoma G361 +

About 2 μg polyA RNA from each of the 8 cell lines indicated in Table 1above were placed on each lane of a denaturing formaldehyde 1.2% agarosegel, separated by denaturing gel electrophoresis to ensure intactness,transferred to a charge-modified nylon membrane by Northern blotting,and fixed by UV irradiation. Lanes 1 to 8 contained RNA frompromyelocytic leukemia HL-60, HeLa cell line S3, human chronicmyelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt'slymphoma Raji, colorectal adenocarcinoma SW 480, lung carcinoma A549 andmelanoma G361, respectively. RNA size marker lines at 9.5, 7.5, 4.4,2.4, and 1.35 kb were indicated in ink on the left margin of the blot,and utilized as sizing standards, and a notch was cut out from the lowerleft hand corner of the membrane to provide orientation. Radiolabeledhuman β-actin cDNA was utilized as a control probe for matching ofdifferent batches of polyA RNAs. A single control band at 2.0 kb in alllanes spotted is confirmatory.

The blots were probed with the full length rat PTTG cDNA probe (SEQ. IDNo: 1; 974 bp) at 60° C. for 1 hr. in ExpressHyb hybridization solution(Clontech) as described by Sambrook et al., the relevant section ofwhich reference is incorporated herein by reference. See, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd. Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The blots were thenwashed twice for 15 min at room temperature in 2×SSC, 0.05% SDS, andtwice for 15 min at 50° C. in 0.1% SSC, 0.1% SDS. A more detaileddescription of the remaining experimental procedures masy be found inPei & Melmed, the relevant section of which is incorporated herein byreference. See, Pei & Melmed, Endocrinology 4: 433-441 (1997).

All cells tested by the Northern blot analysis as described aboveevidenced expression of human PTTG, including lymphoma, leukemia,melanoma and lung carcinomas, among others.

Example 10

Cloning of Human PTTG cDNA

A human fetal liver cDNA library (Clontech, Palo Alto, Calif.) wasscreened as described by Maniatis et al, using a radioactively labeledcDNA fragment of the entire rat PTTG coding region as a probe. See,Maniatis et al, Molecular cloning, Cold Spring Harbor Press, 1989. ThecDNA inserts from positive clones were subcloned into plasmidpBluescript-SK (Stratagene, La Jolla, Calif.), and subjected to sequenceanalysis using Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio).The sequence of the nucleic acid is provided in Table 5 below.

TABLE 5 PTTG Nucleic Acid Sequences   1 ATGGCCGCGA GTTGTGGTTT AAACCAGGAGTGCCGCGCGT CCGTTCACCG  51 CGGCCTCAGA TGAATGCGGC TGTTAAGACC TGCAATAATCCAGAATG GCT 101 ACT CTG ATC TAT GTT GAT AAG GAA AAT GGA GAA CCA GGC ACC123 CGT GTG GTT GCT AAG GAT GGG CTG AAG CTG GGG TCT GGA CCT 185TCA ATC AAA GCC TTA GAT GGG AGA TCT CAA GTT TCA ACA CCA 207CGT TTT GGC AAA ACG TTC CAT GCC CCA CCA GCC TTA CCT AAA 249GCT ACT AGA AAG GCT TTG GGA ACT GTC AAC AGA GCT ACA GAA 311AAG TCT GTA AAG ACC AAG GGA CCC CTC AAA CAA AAA CAG CCA 350CCA AGC TTT TCT GCC AAA AAG ATG ACT GAG AAG ACT GTT AAA 392GCA AAA AGC TCT GTT CCT GCC TCA GAT GAT GCC TAT CCA GAA 334ATA GAA AAA TTC TTT CCC TTC AAT CCT CTA GAC TTT GAG AGT 376TTT GAC CTG CCT GAA GAG CAC CAG ATT GCG CAC CTC CCC TTG 419AGT GGA GTG CCT CTC ATG ATC CTT GAC GAG GAG AGA GAG CTT 560GAA AAG CTG TTT CAG CTG GGC CCC CCT TCA CCT GTG AAG ATG 602CCC TCT CCA CCA TGG GAA TCC AAT CTG TTG CAG TCT CCT TCA 644AGC ATT CTG TCG ACC CTG GAT GTT GAA TTG CCA CCT GTT TGC 686TGT GAC ATA GAT ATT TAA 704     ATTTCTT AGTGCTTCAG AGTTTGTGTG TATTTGTATTAATAAAGCAT 751 TCTTTAACAG ATAAAAAAAA AAAAAAAAA (SEQ. ID No:3)

A complete open reading frame containing 606 bp was found in thepositive clones. The homology between the nucleotide sequences of theopen reading frame and the coding region of rat PTTG or PTSG (oldnomenclature) is 85%. The deduced amino acid sequence is shown in Table6 below.

TABLE 6 PTTG Aminoe Acid Sequence   1 MATLIYVDKE NGEPGTRVVA KDGLKLGSGPSIKALDGRSQ VSTPRFGKTF  51 DAPPALPKAT RKALGTVNRA TEKSVKTKGP LKQKQPSFSAKKMTEKTVKA 101 KSSVPASDDA YPEIEKFFPF NPLDFESFDL PEEHQIAHLP LSGVPLMILD151 EERELEKLFQ LGPPSPVKMP SPPWESNLLQ SPSSILSTLD VELPPVCCDI 201 DI* (SEQID NO:4)

A comparison of the amino acid sequence the human PTTG translatedproduct of this open reading frame and that of the rat PTTG proteinreveals 77% identity and 89% homology. The cDNAs obtained from theseclones represents human homologies of rat PTTG. No other cDNA fragmentswith higher homology were detected from the library.

Example 11

Tissue Distribution of Human PTTG mRNA

Total RNA was prepared using Trizol Reagent (Gibco-BRL, Gaithersburg,Md.) from normal human pituitary glands (Zoion Research Inc. Worcester,Mass.) and fresh human pituitary tumors collected at surgery and frozenin liquid nitrogen. 20 mg total RNA were used for 1% agarose gelelectrophoresis. RNA blots (Clontech, Palo Alto, Calif.) derived fromnormal adult and fetal tissues as well as from malignant tumor celllines, were hybridized with radioactively labeled human cDNA fragmentcontaining the complete coding region. The RNA isolated from each cellline was transferred onto a nylon membrane (Amersham, Arlington Heights,Ill.), and hybridized with radioactively labeled probe at 55° C.overnight in 6×SSC, 2×Denhardt's solution, 0.25% SDS. The membranes werewashed twice at room temperature for 15 minutes each, and then for 20minutes at 60° C. in 0.5×SSC, 0.1% SDS, and autoradiographed. Theautoradiography was carried out using Kodak BIOMEX-MR film (EastmanKodak, Rochester, N.Y.) with an intensifying screen. The blots werestripped by washing for 20 minutes in distilled water at 95° C. forsubsequent probing.

The results from the Northern blot analysis indicated that PTTG isexpressed in liver, but not in brain, lung, and kidney of human fetaltissue. In addition, PTTG is strongly expressed in testis, modestlyexpressed in thymus, and weakly expressed in colon and small intestineof normasl human adult tissue. No expression was detected by Northernanalysis in brain, heart, liver, lung, muscle, ovary, placenta, kidney,and pancreas.

The expression of PTTG in several human carcinoma cell lines was alsoanalyzed by Northern blots. In every carcinoma cells examined, PTTG wasfound highly expressed. The human tumor cell lines tested are listed inTable 7 below.

TABLE 7 Tested Human Tumor Cell Lines Promyelocytic leukemia HL-60Epitheloid carcinoma HeLa cell S3 Chronic myelogenous leukemia K-562Lymphoblastic leukemia MOLT-4 Burkitt's lymphoma Raji Colorectaladenocarcinoma SW 480 Lung carcinoma A549 Melanoma G361 Hepatocellularcarcinoma Hep 3B Thyroid carcinoma TC-1 Breast adenocarcinoma MCF-7Osteogenic sarcoma U2 OS Placenta choriocarcinoma JAR ChoriocarcinomaJEG-3

Example 12

Human PTTG Expression in Normal Pituitary and Pituitary Tumors

RT-PCR was performed as follows. 5 μg total RNA were treated with 100 URNase-free DNase I at room temperature for 15 minutes. DNase I wasinactivated by incubation at 65° C. for 15 minutes. The sample was thenused for reverse transcription using oligo-dT primer and SuperScript IIreverse transcriptase (Gibco-BRL, Gaithersburg, Md.). After reversetranscription, the sample was subjected to PCR amplification with PCRSuperMix (Gibco-BRL, Gaithersburg, Md.) using hPTTG-specific primers andhuman cyclophilin A-specific primers as an internal control.

Northern blot analysis indicated that the level of expression of PTTG isquite low in normal pituitary as well as in pituitary tumors. Therefore,comparative RT-PCR was used to study the expression of PTTGquantitatively in normal pituitary and pituitary tumors. The results ofthis study showed that in most of pituitary tumors tested, includingnon-functioning tumors, GH-secreting tumors, and prolactinomas, theexpression level of PTTG was higher than that of normal pituitary.

Example 13

Stable Transfection of Human PTTG into NIH 3T3 Cells

The complete coding region of hPTTG cDNA was subcloned in reading frameinto the mammalian expression vector pBK-CMV (Stratagene, La Jolla,Calif.), and transfected into NIH 3T3 fibroblast cells by Lipofectamine(Gibco-BRL, Gaithersburg, Md.) according to manufacturer's protocol. 24hours after transfection, the cells were serially diluted and grown inselection medium containing 1 mg/ml G418 for 2 weeks. Individual cloneswere isolated and maintained in selection medium. Total RNA was isolatedfrom hPTTG-transfected cell lines as well as from control cells in whichblank vector pBK-CMV had been transfected. Northern blot was performedto confirm overexpression of hPTTG in transfected cell lines. These celllines were used in subsequent cell proliferation assay as well as invitro and in vivo transformation assay.

Example 14

Cell Proliferation Assay

A cell proliferation assay was performed using the CellTiter 96non-radioactive cell proliferation assay kit (Promega Medicine, Wis.)according to the manufacturer's protocol. 5,000 cells were seeded in96-well plates and incubated at 37° C. for 24-72 hours. Eight wells wereused for each clone in each assay. At each time point, 15 μl of dyesolution was added to each well and the cells were incubated at 37° C.for 4 hours. After incubation, 100 μl solubilization/stop solution wereadded to each well, and the plates incubated overnight at roomtemperature. The absorbance was determined at 595 nm using an ELISAplate reader.

Control and hPTTG-overexpressing NIH 3T3 cells were used to perform thisassay. The results indicated that the growth of cells transfected withthe PTTG-expressing vector was suppressed by 30˜45% as compared withcells transfected with blank vector. These results clearly show that thePTTG protein inhibits cell proliferation.

Example 15

In vitro and In vivo Transformation Assay

(a) In vitro Transformation Assay

Control and hPTTG-transfected cells were tested foranchorage-independent growth in soft agar. 3 ml of soft agar (20% of2×DMEM, 50% DMEM, 10% fetal bovine serum, and 20% of 2.5% agar, meltedand mixed at 45° C.) were added to 35mm tissue dishes. 10,000 cells weremixed with 1 ml soft agar and added to each dish, and iincubated for 2weeks until colonies could be counted and photographed.

(b) In vivo Transformation Assay

5×10⁵ cells containing either a blank vector or hPTTG-expressing cellswere injected into nude mice. The mice were sacrificed two weeks afterinjection, and the tumors formed near the injection sites examined.

When the NIH 3T3 cells stably transfected with the PTTG-expressingvector were tested in an anchorage-independent growth assay, these cellscaused large colony formation on soft agar, suggesting the transformingability of PTTG protein.

When the NIH 3T3 cells were injected into nude mice, they caused in vivotumor formation within 2 weeks after injection. These data indicate thathuman PTTG, as its rat homologue, is a potent transforming gene.

Summary of Sequences

SEQ ID NO:1 is the nucleic acid sequence (and the deduced amino acidsequence) of cDNA encoding the rat PTTG protein of the presentinvention.

SEQ ID NO:2 is the deduced amino acid sequence of the rat PTTG proteinof the present invention.

SEQ. ID No:3 is the nucleic acid sequence of cDNA encoding a human PTTGprotein of this invention.

SEQ. ID No:4 is the deduced amino acid sequence of a human PTTG proteinof this invention.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it should be understood thatmodifications and variations to the embodiments and exemplary disclosureprovided, are within the spirit and scope of the invention as describedand claimed in this patent.

7 1 974 DNA Rattus rattus 1 aattcggcac gagccaacct tgagcatctg atcctcttggcttctccttc ctatcgctga 60 gctggtaggc tggagacagt tgtttgggtg ccaacatcaacaaacgattt ctgtagttta 120 gcgtttatga ccctggcgtg aagatttaag gtctggattaagcctgttga cttctccagc 180 tacttctaaa tttttgtgca taggtgctct ggtctctgttgctgcttagt tcttccagcc 240 ttcctcaatg ccagttttat aatatgcagg tctctcccctcagtaatcca ggatggctac 300 tctgatcttt gttgataagg ataacgaaga gccaggcagccgtttggcat ctaaggatgg 360 attgaagctg ggctctggtg tcaaagcctt agatgggaaattgcaggttt caacgccacg 420 agtcggcaaa gtgttcggtg ccccaggctt gcctaaagccagcaggaagg ctctgggaac 480 tgtcaacaga gttactgaaa agccagtgaa gagtagtaaacccctgcaat cgaaacagcc 540 gactctgagt gtgaaaaaga tcaccgagaa gtctactaagacacaaggct ctgctcctgc 600 tcctgatgat gcctacccag aaatagaaaa gttcttccccttcgatcctc tagattttga 660 gagttttgac ctgcctgaag agcaccagat ctcacttctccccttgaatg gagtgcctct 720 catgatcctg aatgaagaga gggggcttga gaagctgctgcacctggacc ccccttcccc 780 tctgcagaag cccttcctac cgtgggaatc tgatccgttgccgtctcctc ccagcgccct 840 ctccgctctg gatgttgaat tgccgcctgt ttgttacgatgcagatattt aaacgtctta 900 ctcctttata gtttatgtaa gttgtattaa taaagcatttgtgtgtaaaa aaaaaaaaaa 960 aaactcgaga gtac 974 2 199 PRT Rattus rattus 2Met Ala Thr Leu Ile Phe Val Asp Lys Asp Asn Glu Glu Pro Gly Ser 1 5 1015 Arg Leu Ala Ser Lys Asp Gly Leu Lys Leu Gly Ser Gly Val Lys Ala 20 2530 Leu Asp Gly Lys Leu Gln Val Ser Thr Pro Arg Val Gly Lys Val Phe 35 4045 Gly Ala Pro Gly Leu Pro Lys Ala Ser Arg Lys Ala Leu Gly Thr Val 50 5560 Asn Arg Val Thr Glu Lys Pro Val Lys Ser Ser Lys Pro Leu Gln Ser 65 7075 80 Lys Gln Pro Thr Leu Ser Val Lys Lys Ile Thr Glu Lys Ser Thr Lys 8590 95 Thr Gln Gly Ser Ala Pro Ala Pro Asp Asp Ala Tyr Pro Glu Ile Glu100 105 110 Lys Phe Phe Pro Phe Asp Pro Leu Asp Phe Glu Ser Phe Asp LeuPro 115 120 125 Glu Glu His Gln Ile Ser Leu Leu Pro Leu Asn Gly Val ProLeu Met 130 135 140 Ile Leu Asn Glu Glu Arg Gly Leu Glu Lys Leu Leu HisLeu Asp Pro 145 150 155 160 Pro Ser Pro Leu Gln Lys Pro Phe Leu Pro TrpGlu Ser Asp Pro Leu 165 170 175 Pro Ser Pro Pro Ser Ala Leu Ser Ala LeuAsp Val Glu Leu Pro Pro 180 185 190 Val Cys Tyr Asp Ala Asp Ile 195 3779 DNA Homo sapiens 3 atggccgcga gttgtggttt aaaccaggag tgccgcgcgtccgttcaccg cggcctcaga 60 tgaatgcggc tgttaagacc tgcaataatc cagaatggctactctgatct atgttgataa 120 ggaaaatgga gaaccaggca cccgtgtggt tgctaaggatgggctgaagc tggggtctgg 180 accttcaatc aaagccttag atgggagatc tcaagtttcaacaccacgtt ttggcaaaac 240 gttcgatgcc ccaccagcct tacctaaagc tactagaaaggctttgggaa ctgtcaacag 300 agctacagaa aagtctgtaa agaccaaggg acccctcaaacaaaaacagc caagcttttc 360 tgccaaaaag atgactgaga agactgttaa agcaaaaagctctgttcctg cctcagatga 420 tgcctatcca gaaatagaaa aattctttcc cttcaatcctctagactttg agagttttga 480 cctgcctgaa gagcaccaga ttgcgcacct ccccttgagtggagtgcctc tcatgatcct 540 tgacgaggag agagagcttg aaaagctgtt tcagctgggccccccttcac ctgtgaagat 600 gccctctcca ccatgggaat ccaatctgtt gcagtctccttcaagcattc tgtcgaccct 660 ggatgttgaa ttgccacctg tttgctgtga catagatatttaaatttctt agtgcttcag 720 agtttgtgtg tatttgtatt aataaagcat tctttaacagataaaaaaaa aaaaaaaaa 779 4 202 PRT Homo sapiens 4 Met Ala Thr Leu IleTyr Val Asp Lys Glu Asn Gly Glu Pro Gly Thr 1 5 10 15 Arg Val Val AlaLys Asp Gly Leu Lys Leu Gly Ser Gly Pro Ser Ile 20 25 30 Lys Ala Leu AspGly Arg Ser Gln Val Ser Thr Pro Arg Phe Gly Lys 35 40 45 Thr Phe Asp AlaPro Pro Ala Leu Pro Lys Ala Thr Arg Lys Ala Leu 50 55 60 Gly Thr Val AsnArg Ala Thr Glu Lys Ser Val Lys Thr Lys Gly Pro 65 70 75 80 Leu Lys GlnLys Gln Pro Ser Phe Ser Ala Lys Lys Met Thr Glu Lys 85 90 95 Thr Val LysAla Lys Ser Ser Val Pro Ala Ser Asp Asp Ala Tyr Pro 100 105 110 Glu IleGlu Lys Phe Phe Pro Phe Asn Pro Leu Asp Phe Glu Ser Phe 115 120 125 AspLeu Pro Glu Glu His Gln Ile Ala His Leu Pro Leu Ser Gly Val 130 135 140Pro Leu Met Ile Leu Asp Glu Glu Arg Glu Leu Glu Lys Leu Phe Gln 145 150155 160 Leu Gly Pro Pro Ser Pro Val Lys Met Pro Ser Pro Pro Trp Glu Ser165 170 175 Asn Leu Leu Gln Ser Pro Ser Ser Ile Leu Ser Thr Leu Asp ValGlu 180 185 190 Leu Pro Pro Val Cys Cys Asp Ile Asp Ile 195 200 5 16 DNAHomo sapiens 5 aagctttttt tttttg 16 6 13 DNA Homo sapiens 6 aagcttgctgctc 13 7 16 DNA Homo sapiens n = a, g, or c 7 aagctttttt tttttn 16

What is claimed is:
 1. An isolated polynucleotide, comprising a PTTGcoding region of SEQ ID No: 3, or a nucleic acid fully complementarythereto.
 2. The polynucleotide of claim 1, which is an RNA.
 3. Thepolynucleotide of claim 1, which is a DNA.
 4. A vector comprising thepolynucleotide of claim
 1. 5. A composition, comprising the vector ofclaim 4, and a carrier.
 6. A host cell carrying the vector of claim 4.7. A recombinant host cell carrying the polynucleotide of claim
 1. 8. Acomposition, comprising the host cell of claim 7, and a carrier.
 9. Anisolated polynucleotide, comprising a nucleic acid encoding apolypeptide having an amino acid sequence of SEQ ID NO: 4, or a nucleicacid fully complementary thereto.
 10. The polynucleotide of claim 9,which is an RNA.
 11. The polynucleotide of claim 9, which is a DNA. 12.A vector comprising the polynucleotide of claim
 9. 13. A composition,comprising the vector of claim 12, and a carrier.
 14. A host cellcarrying the vector of claim
 12. 15. A composition, comprising the hostcell of claim 14, and a carrier.
 16. A recombinant host cell carryingthe polynucleotide of claim 9.